The Escherichia coli gene encoding one of the primosomal proteins, protein i, was cloned by the use of synthetic oligonucleotide probes. Nucleotide sequence analysis revealed a coding region for protein i of 537 base pairs preceded by a possible promoter sequence. The gene is located adjacent to the dnaC locus, probably both being in a single operon. The protein i gene was shown to be closely related to the dnaT locus based on the following observations. (i) A multicopy plasmid carrying only the protein i gene suppresses the temperature-sensitive phenotype of a dnaT strain and restores the ability of the strain to carry out stable DNA replication in the absence of protein synthesis. (ii) An extract from a dnaT strain does not support replication of the plasmid pBR322 in vitro; addition of purified protein i restores its activity. These results indicate that protein i is encoded by dnaT and that it is essential for chromosomal DNA replication and is involved in the induction of stable DNA replication during the SOS response.